Method of regeneration of Medicago sativa and expressing foreign DNA in same

ABSTRACT

The invention relates to improved transformation and regeneration of alfalfa,  Medicago sativa.    
     Regeneration and transformation of alfalfa is made possible by use of immature cotyledons of alfalfa. By using immature cotyledon tissue, it is possible to regenerate and transform varieties of alfalfa never before capable of regeneration and transformation.

This application is a continuation-in-part of previously filed andco-pending application U.S. Ser. No. 09/003,178, filed Jan. 5, 1998,which is a continuation of application Ser. No. 08/497,597 filed Jun.30, 1995, now U.S. Pat. No. 5,731,202, which is a continuation of U.S.Ser. No. 08/386,139, filed Feb. 9, 1995, now abandoned, which is acontinuation of U.S. Ser. No. 08/213,278 filed Mar. 15, 1994, nowabandoned, which is a divisional of U.S. Ser. No. 07/817,205 filed Jan.6, 1992.

BACKGROUND OF THE INVENTION

Genetic transformation of plants has been one of the major advancesachieved in biotechnology and its contributions to producing improvedplants, improved crops, and consequently improved availability of foodworldwide has been widely recognized. In certain plants, however,transformation has been especially difficult to achieve, andtransformation of the valuable forage crop alfalfa, Medicago sativa hasbeen inhibited by the peculiarities of the plant.

Transformation of alfalfa has been hampered primarily by two majorlimitations: constraints imposed by the method of transformation, andthe poor regeneration from tissue and cell cultures of many alfalfavarieties.

The first limitation occurs because alfalfa is presently primarilytransformed through the use of Agrobacterium tumefaciens. Agrobacteriumexhibits host strain specificity and only certain Agrobacterium strainswill infect a few alfalfa genotypes. The ability to transform alfalfa isconsiderably limited as a result. The second major inhibition oftransformation of alfalfa is its own poor regeneration frequency. Only afew varieties exhibit even modest regeneration, and those elitevarieties providing superior performance in the field are notoriouslypoor regenerators. The combination of these two problems has created aconsiderable bottle-neck in achieving transformation of the plant.

Alfalfa exhibits other traits setting it apart from many crop plants. Itis an autotetraploid and is frequently self incompatible in breeding.When selfed, the pollen may not germinate or, when it does, later stopsgerminating. Thus producing a true breeding parent for hybrids is notpossible, which complicates breeding substantially. It has beendetermined that there are nine major germ-plasma sources of alfalfa: M.falcata, Ladak, M. varia, Turkistan, Flemish, Chilean, Peruvian, Indian,and African.

Culture of explant source tissue, such as mature cotyledons andhypocotyls, demonstrates the regeneration frequency of genotypes in mostcultivars is only about ten percent. SeitzKris, M. H. and E. T. Bingham,In vitro Cellular and Developmental Biology 24 (10): 1047-1052 (1988).Efforts have been underway to improve regeneration, and have includedattempts at asexual propagation to maintain individual genotypes whichpossess the regeneration trait. Further, propagation by asexual methodsis not practical if many genotypes are involved. Bingham and others haveattempted to avoid this problem by recurrent selection. In the firstcycle, regenerating genotypes were selected, crossed and recycled untilregeneration was improved to 60 percent or better. The result of thiswas the development of Regen-S, in which two-thirds of the plants werecapable of regeneration from callus tissue. E. T. Bingham, et. al., CropScience 15:719-721 (1975).

Additionally, researchers believe that somatic embryogenesis in alfalfais inheritable, and is controlled by relatively few genes. Efforts atimproving regeneration have thus been directed towards isolation of thegenetic control of embryogenesis, and breeding programs which wouldincorporate such information. See, e.g. M. M. Hernandez-Fernandez, andB. R. Christie, Genome 32:318-321 (1989); I. M. Ray and E. T. Bingham,Science 29:1545-1548 (1989). This is complicated by the characteristicsof alfalfa noted above.

This invention relates to improvements in transformation andregeneration of alfalfa by departing from these previous approaches. Inone embodiment, direct introduction of DNA is accomplished by the use ofmicroprojectile bombardment. As a result of the use of bombardment, thelimitations of Agrobacterium are overcome.

Furthermore, limitations in regeneration of alfalfa are overcome byselecting immature cotyledons for transformation and regeneration. Ithas been found that when immature cotyledons of alfalfa are used,regeneration is considerably improved, and there are no limitations onwhat type of alfalfa can be regenerated as a result of this method. Thuseven elite varieties may be regenerated, and transformed and there is nolonger a limitation imposed by the method of transformation used, orvariety transformed.

Thus, it is an object of this invention to improve transformation ratesof Medicago sativa.

It is another object of this invention to improve regeneration ofMedicago sativa. A still further object of this invention is to allowtransformation and regeneration of any variety of Medicago sativa.

Still further objects of the invention will become apparent through thefollowing description.

SUMMARY OF THE INVENTION

Microprojectile bombardment is used to transform DNA into Medicagosativa, resulting in introduction of DNA into any variety of Medicagosativa. The invention further relates to the use of immature cotyledonsof Medicago sativa for transformation and regeneration of any variety ofMedicago sativa.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map of plasmid pPHI251.

FIG. 2 is a map of plasmid pPHI256.

FIG. 3 is a graph showing time course harvest results plotting age ofthe cotyledon on the x axis and regeneration response on the y axis.

FIG. 4 is a graph of regeneration using immature cotyledons (clear bar)and mature cotyledons (solid bar) of varieties listed.

FIG. 5 is a map of plasmid pPHI413.

DESCRIPTION OF THE INVENTION

The inventors have discovered that by use of the tissue of immaturecotyledons of alfalfa, any transformation method may be usedsuccessfully, and regeneration achieved, and the process is not limitedto non-elite alfalfa. By elite alfalfa, the inventors refer to thosevarieties or genotypes used commercially or commercially desirable butwhich prior to the invention were not capable of consistent reliabletransformation and regeneration. The following is set forth toillustrate various embodiments of the invention and is not intended tolimit its scope.

Microprojectile Bombardment

Microprojectile bombardment in order to transform plant cells is knownto those skilled in the art. The general process has been described byT. M. Klein, et al. Proc. Natl. Acad. Sci. USA 85:4305-4309 (1988) anddiscussed at Sanfor et al. U.S. Pat. No. 4,945,050. These references, aswell as those cited throughout, represent knowledge of those skilled inthe art and are each incorporated herein by reference. The basic processincludes coating DNA onto small high density particles,microprojectiles, which are then placed into the particle gun or heliumgun apparatus and accelerated to a high velocity in order to penetrateplant and carry the DNA or other substance into the interior of thebombarded cell.

Klein describes his work as follows: His report describes a process ofdelivering foreign genes into maize cells that does not require theremoval of cell walls and is capable of delivering DNA into embryogenicand nonembryogenic tissues. A plasmid harboring a chimericchloramphenicol acetyltransferase (CAT) gene was absorbed to the surfaceof microscopic tungsten particles (microprojectiles). Thesemicroprojectiles were then accelerated to velocities sufficient forpenetrating the cell walls and membranes of maize cells in suspensionculture. High levels of CAT activity were consistently observed afterbombardment of cell cultures of the cultivar Black Mexican Sweet, whichwere comparable to CAT levels observed after electroporation ofprotoplasts. Measurable increase in CAT levels were also observed in twoembryogenic cell lines after bombardment. Gene expression was observedonly when an intron from the alcohol dehydrogenase 1 gene of maize wasligated between the 35S promoter and the CAT coding region. CAT activitywas detected in cell cultures bombarded with microprojectiles with anaverage diameter of 1.2 um, but not after bombardment withmicroprojectiles 0.6 or 2.4 um in diameter. Bombarding the same sampleseveral times was found to markedly enhance CAT activity. These resultsdemonstrate that the particle bombardment process can be used to deliverforeign DNA into intact cells of maize.

The plasmids which were used in Klein's study have been described atCallis, J., Fromm, M. and Walbot, V. (1987) Genes Dev. 1. 1183-1200. Theplasmid pCaMVIICN consists of the 35S promoter from cauliflower mosaicvirus, a fragment (Bcl I/BamHI) from the alcohol dehydrogenase intron 1(Adh 1), a CAT coding region, and the nopaline synthase polyadenylationregion. The plasmid pCaMVCN is identical to pCaMVICN but lacks the Adhlintron fragment.

Embryogenic suspension cultures 1-86-17 and 13-217 were derived fromtype II embryogenic callus (Green, C. E., Armstrong, C. L. & Anderson,P. C. (1983) in Molecular Genetics of Plants and-Animals, ads. Downey,K., Voellmy, R. M., Fazelahmad, A. & Schultz, J. (Academic, New York),Vol. 20, pp. 147). The callus was initiated from a maize inbreddesignated R21 (for line 3-86-17) or B73×G35 (for line 13-217). InbredR21 was derived from a regenerated plant from a long-term callus cultureof public inbred B73 and is very similar to B73. Both R21 and G35 areproprietary inbred lines of Pioneer Hi-Bred International (Johnston,IA). Suspension cultures of the cultivar BMS were obtained from V.Walbot (Stanford University). Suspension cultures were maintained inMurashige and Skoog (MS) medium (Murashige, T. & Skoog, F. (1962)Physiol. Plant 15, 473497) supplemented with 2,4-dinitrophenol (2mg/liter) and sucrose (30 g/liter). (See further discussion below). Thesuspension cultures were passed through a 710-um sieve 7 days prior tothe experiment, and the filtrate was maintained in MS medium. Inpreparation for bombardment with the microprojectiles, cells wereharvested from suspension culture by vacuum filtration on a Buchnerfunnel (Whatman no. 614). The same cell batch from each genotype wasused within each experiment.

The particle gun device and general methods for bombardment of cellswith microprojectiles have been described (Sanford, J. C., Klein, T. M.,Wolf, E. D. & Allen, N. (1987) Particle Sci. Technol., 5, 27-37 andKlein T. M., Wolf, E. D., Wu, R. & Sanford, J. C. (1987) Nature (London327, 70-73). Before bombardment, cells (100 mg fresh weight) were placedin a 3.3-cm Petri dish. The cells were dispersed in 0.5 ml of freshculture medium to form a thin layer of cells covered by a film of mediumThe uncovered Petri dish was placed in the sample chamber and a vacuumpump was used to decrease the pressure in the chamber to 0.1 atm (1atm=101.3 kPa) (operation in a partial vacuum allows themicroprojectiles to maintain their velocity over a longer distance,since air resistance is an important factor in their deceleration).

Cells were bombarded with tungsten particles with an average diameter of1.2 um (GTE Sylvania). In one experiment, cells were also bombarded withmicroprojectiles with an average diameter of 0.6 um (GTE Sylvania) or2.4 um (General Electric). Plasmid DNA was absorbed to themicroprojectiles by adding 5 ul of DNA (1 ug per ul of TE buffer, pH7.7; 0.1 M) to 25 ul of a suspension of tungsten particles (0.05 g perml of distilled water) in a 1.5-ml Eppendorf tube. CaCl₂ (25 ul of 2.5 Msolution and spermidine free base (10 ul of 0.1 M solution) were thenadded to the suspension. Particles became agglomerated and settled tothe bottom of the Eppendorf tube=10 min after addition of CaCl₂ andspermidine. Most of the supernatant (45 ul) was then removed and theparticles were deagglomerated by briefly (1 sec) touching the outside ofthe Eppendorf tube to the probe (hom type) of a sonicator (Heat SystemUltrasonics, Plainview, N.Y.). Five microliters of the resultingsuspension of microprojectiles was then placed on the front surface of acylindrical polyethylene macroprojectile. The macroprojectile was thenplaced into the barrel of the particle gun device and a blank gun powdercharge (no. 1 gray extra light; Speed Fasteners, Saint Louis) was loadedinto the barrel behind the macroprojectile. A firing pin device was usedto detonate the gun powder charge, accelerating the macroprojectile downthe barrel of the device where it impacts with a stopping plate. Uponimpact, the microprojectiles are propelled from the front surface of themacroprojectile and continue toward the cells through a small aperturein the stopping plate. The cells were positioned 15 cm from the end ofthe barrel of the particle gun. After bombardment, the Petri dish wasremoved from the apparatus and the cells were transferred to 5 ml offresh medium in a 1 5-ml polypropylene tube. The cells were thenmaintained in this tube with agitation at 27° C. until harvested foranalysis. Controls consisted of cells bombarded with microprojectileslacking DNA.

In one set of experiments, the cells were treated either with a mediumof high osmotic potential or with a mixture of medium an mineral oilduring bombardment. In the first case, 100 mg of BMS cells weredispersed in 0.5 ml of MS medium supplemented with mannitol (0-4 M), 30min prior to bombardment. After particle bombardment, the cells wereleft in the mannitol-containing medium for an additional 30 min, afterwhich they were resuspended in 5 ml of standard MS medium. In the secondcase, the cells were dispersed in an emulsion of either 0.1 ml ofsterile mineral oil and 0.5 ml of MS medium, 0.2 ml of mineral oil and0.4 ml of MS medium, or 0.6 ml of MS medium lacking mineral oil. Afterbombardment, the cells were suspended in 5 ml of MS medium.

Analyses of CAT activity were performed 96 hours after bombardment.Tissue extracts were prepared by sedimenting the cells at=13,000×g for10 min in a 1.5-mi Microfuge tube. The supernatant was removed and 100ul of buffer (0.25 M Tds-HCI, pH 7.8) was added to the pellet. Thesample was homogenized on ice for about 2 minutes with a disposablepolypropylene pestle (Kontes) driver at 300 rpm by an electric motor.After grinding, the sample was Vortex mixed briefly to complete theextraction of soluble protein. Cell debris was removed by centrifugationat 13,000×g in a Microfuge at 40C for 10 min. The supernatant wasdecanted and normalized to a volume of 200 ul with the Tds-HCI buffer.

The CAT activity in the extracts was determined as reported (Gorman, C.M. , Moffat, L. F. & Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051)except the samples were heated at 60° C. for 12 min before addition ofsubstrates. The reaction mixture was incubated for 1. 5 hr at 37° C.,and reaction products were extracted from the mixture with 30 ul of coldethyl acetate, air dried, and resuspended in 20 ul of ethyl acetate forspotting on TLC plates (Baker, Phillipsburg, N.J.). After TLC resolutionof chloramphenicol and its acetylated derivatives by usingchlorofomi/methanai (95:5), autoradiograms of the TLC plates were made(60-hr exposure at 22° C.; DuPont Cronex film) quantitative results wereobtained by scintillation counting of separated spots of chloramphenicoland its acetylated derivatives, and the percentage conversion toacetylated products was calculated. CAT activity (1 unit of CAT)catalyzes the acetylation of 1 ng of chloramphenicol per min at 37° C.was determined by comparison with a standard curve of acetylationconversions obtained with purified bacterial CAT (P-L Biochemicals).Protein in the cell extracts was determined according to Bradford(Bradford, M. M. (1976) Anal. Biochem. 72, 248-254). CAT activity wasstandardized on the basis of units of CAT activity per mg of solubleprotein.

Obviously, there may be numerous modifications to this basic process,but Klein describes at least one method of accomplishing microprojectilebombardment.

Previous work involved delivery of foreign genes through this methodinto intact plant of tobacco tissue, but its application to theeconomically important species alfalfa has not been successfullyaccomplished. Tomes, et al. Plant Molecular Biology 14:261-268 (1990).Microprojectile bombardment of alfalfa to achieve transformation has notbeen previously reported.

Introduction of DNA into a plant is demonstrated at first by transientexpression. Short term expression is noted by confirming the presence ofthe DNA within the plant cells 24 to 48 hours after bombardment. Whenexpressed up to 72 hours after bombardment it is demonstrated that theDNA has been delivered via the particle gun or other method and that theDNA vector functions. When continuing to be expressed two to eight weeksafter bombardment, it may be concluded the DNA is persistent and likelyintegrated into the plant genome. Its ability to persist at this pointshows it has survived attack from nucleases which typically would attackunprotected foreign DNA. When the R₀ plants are recovered, continuingexpression is further indication that stable transformation into theplant cells has occurred. Southern analysis allows confirmation of this.When crossed and the R₁ generation analyzed, expression andinheritibility of the DNA is further confirmed.

A variety of plant cell sources can be used for transformation bymicroprojectile bombardment. Hypocotyls, cotyledons of mature seed andpetioles are plant tissue which can be subjected to bombardment. Theapplicant has discovered that when cotyledons are used, satisfactorytransformation results. While not wishing to be bound by any theory, itis proposed that cotyledons may be a better source of tissue forbombardment because the cells to be bombarded are those which arecapable of giving rise to plants. Mature cotyledons are also convenientsources of tissue and easy to excise from the seed.

Cotyledons from mature seed can be used in transformation, that is, seedwhich has reached dormancy. This seed is then placed in water, typicallyfor one to several days, the root breaks through the seed coat, and thecotyledon is dissected. The use of immature cotyledons is discussed morefully below.

It has been found that the optimum stage for best transformation resultsof mature cotyledons occurs when bombarded after 24 to 120 hours ofimbibing water. It has been discovered that at this point regeneration,transient transformation, and resulting transformation is at itsoptimum. Prior to 24 hours it is as a practical matter more difficult toremove the seed coat without damaging the cotyledon. After 120 hours, itis more difficult to regenerate the plants.

The tissue should be bombarded one or two times, and bombardments inexcess of this would likely kill the cells. Tissue culture was alsooptimized for the maximum regeneration possibilities. In the experimentsdescribed below, Regen-S, was used. As noted supra, Regen-S is known forits improved regeneration potential. Set forth below are tissue cultureswhich were employed. The most important factor in tissue cultureoptimized for regeneration is high concentration of2,4-dichlorophenoxyacetic acid (2,4-D) as compared to a lowconcentration of kinetin. Tissue/organ culture is described generally byAtanassov and Brown in Plant Cell Tissue Organ Culture 4:111-122 (1985).

Agrobacterium Transformation

As has been noted, until the present invention, the only methodavailable for transformation of alfalfa with heterologous DNA wasthrough the Agrobacterium tumefaciens system. Even then, the onlyvarieties of alfalfa which could be transformed were select non-elitevarieties. With use of the tissue described herein, transformation byuse of Agrobacterium is possible without host strain specificity, thusgreatly broadening the ability to transform the plant.

Agrobacterium tumefaciens is the etiologic agent of crown gall. The wildtype form of Agrobacterium tumefaciens carries the Ti (tumor-inducing)plasmid that directs the production of tumorigenic crown gall growth onthe host plants. The crown gall is produced following the transfer ofthe tumor inducing T-DNA region from the Ti plasmid into the genome ofan infected plant. This DNA-fragment encodes genes for auxin andcytokinin biosynthesis, and it is these hormones in high concentrationthat promote growth of undifferentiated cells in the crown gall.

Transfer of the T-DNA to the plant genome requires that the Tiplasmid-encoded virulence genes as well as the T-DNA borders, a set ofdirect DNA repeats that delineate the region to be transferred. Thevirulence functions of the Agrobacterium tumefaciens host will directthe insertion of the construct into the plant cell DNA when the cell isinfected by the bacteria. See, for example Horsch et al., Science 233:496-498 (1984), and Fraley et al., Proc. Natl Acad. Sci. 80: 4803(1983). The tumor inducing genes can be removed from Ti plasmid vectors,disarming the pathogenic nature of the system, without affecting thetransfer of DNA fragments between the T-DNA borders. Therefore, thetumor inducing genes are generally replaced with a gene encodingresistance to kanamycin, or some other gene, to allow for selection oftransformants, and a gene encoding the desired trait. The Agrobacteriumcontaining the engineered plasmid is co-cultivated with cultured plantcells or wounded tissue. The de-differentiated plant cells are thenpropagated on selective media, and a transgenic plant is subsequentlyregenerated from the transformed cells by altering the levels of auxinand cytokinin in the growth medium.

Co-cultivation of plant tissue with Agrobacterium tumefaciens is widelyused, where the DNA constructs are placed into a binary vector system.Ishida et al., “High Efficiency Transformation of Maize (Zea mays L.)mediated by Agrobacterium tumefaciens” Nature Biotechnology 14:745-750(1996). The binary vector systems divide the Ti plasmid into twocomponents, a shuttle vector and a helper plasmid. The helper plasmid,which is permanently placed in the Agrobacterium host, carries thevirulence genes. However, a much smaller shuttle vector contains T-DNAborders, a broad-host range bacterial origin of replication, antibioticresistance markers, and a multiple cloning site for incorporation of theforeign gene. In the alternative, a similar strategy employsco-integrating Ti plasmid vectors, whereby an intermediate plasmidcontaining antibiotic resistance, the gene to be transferred and oneT-DNA border are used to transform A. tumefaciens containing a disarmedTi plasmid possessing the virulence genes and one T-DNA border. The twoplasmids homologously recombined in vivo at the T-DNA borders placingthe antibiotic resistance gene and the gene of interest between twoT-DNA borders, one from each plasmid. The genes are then transferredinto plant tissue upon co-cultivation.

Since 1986 it has been possible to transform particular varieties ofalflafa using Agrobacterium tumefaciens. Among the numerous referencesdescribing the method is that by Deak et al, “Transformation of Medicagoby Agrobacterium Mediated Gene Transfer” Plant Cell Reports, Spring,1986. The author reports transforming the highly regenerable A2genotype. There the method involves wounding of plant tissue andculturing with a Ti plasmid having T-DNA borders and vir functions witha selectable marker. The method involved the following process. Theco-cultivation method described for protoplasts of Marton et al Nature277:129-130 (1979) was adapted to inoculate stem cuttings of alfalfawith Agrobacterium. Segments of sterile plants 0.5-1.0 cm in length ofMedicago varia genotype A2 were plated into an Erlenmeyer flask (100mlin size) containing 40ml of liquid UM medium and 1 ml of appropriatelydeluted overnight culture of A. tumefaciens strain bo24 (5×10⁷cells/ml). The tissues were incubated on a rotary shaker (150 rpm/min)at 25° C. for three days. The stem segments were then washed three timeswith sterile distilled water and transferred into solid um mediumcontaining Cb, 300 mg/l and Km, 100 mg/l. After three weeks of culturein average 304 green growing spots appeared on each of the treated stemsegments in the presence of Km.

Chabaud, M. et al, have described alfalfa transformation protocols in1988 in “Parameters affecting the frequency of kanamycin resistantalfalfa obtained by Agrobacterium tumefaciens mediated transformation”Plant Cell Reports 7:512-516 (1988). There, the Agrobacteriumtumefaciens strain A281 containing a suitable plasmid (in his experimentit was pVW130) was grown overnight in liquid LB with selectiveantibiotic, at 30° C., and shaken at 300 rpm. The culture was pelletedby centrifugation at 2300×g, for 15 minutes, resuspended in an equalvolume of 0.85% saline and diluted to give 5×10⁷ bacteria/mL. Woundedleaves were dipped briefly into the bacterial suspension and blotted onsterile filter paper. One mL of alfalfa cell suspension was spread oversolidified B5h medium. A filter paper disc was placed on top of the cellsuspension. Dipped tissue was placed on the filter paper and the tissueco-cultivated with bacteria for four days. The tissue was then washed insterile distilled water and blotted. The tissue was placed on solidB5h+carb 300 ug/ml+kan 100 ug/mL. Every three to four weeks the tissuewas transferred to the same fresh selective medium.

The procedure of Desgagnes et al. uses procedures adapted from Chabaudet al. Desgagnes, R. et al “Genetic transformation of commercialbreeding lines of alfalfa (Medicago sativa)” Plant Cell. Tiss. And Org.Cul. 42:129-140 (1995). He used one of three A. tumefaciens strains:A281, LBS4404 or C58, in combination with one of three vectors: pGA643,pBibKan and pGA482. With the procedure he describes, the epicotyls ofthe plants were rooted in MS medium containing 2% sucrose and 0.25mg 1⁻¹IBA (indole-3-butyric acid). The plant tissue was stem-propagated on MSmedia+IBA (0.25 mg 1⁻¹) in Magenta boxes. Natural resistance tokanamycin was determined for each genotype by layering 0.5-cm leaf disksfrom the plants on solid B5h media containing either 0, 10, 25, 50, 100and 200 ug ml⁻¹ kanamycin. Discs were allowed to grow for four weeks.

The inoculum was prepared by inoculating 125-mnl culture flaskscontaining 20 ml LB medium+antibiotics with an overnight sub-culture ofthe desired Agrobacterium strain. Cultures were allowed to grow at 29°C. up to an OD_(600nm) between 0.17 and 0.20. Cells were pelleted at 4°C. by centrifugation at 5000×g and resuspended in an equal volume of0.85% NaCl. Leaf discs (0.5-cm diameter) were cut from sterile plantletsusing a cork-borer. Leaves were immersed in sterile B5h during excisionto prevent excessive drying at the margin of discs. Discs were soaked inthe inoculum during 20 s, blotted on sterile filter paper and layered,abaxial face up, on a co-cultivation set-up consisting of solid B5hwithout antibiotics, covered by 1 ml of a four day liquid alfalfa cellculture in B5h onto which was deposited a filter paper (Whatman #1).After four days of co-cultivation, discs were thoroughly washed withsterile water to remove excess inoculum, blotted and layered onB5h+antibiotics (50 ug ml⁻¹ kanamycin and 300 ug ml⁻¹ carbenicillin).Calluses were allowed to develop at the margin of the disks for 28 daysin B5h+antibiotics.

Calluses were then cut in two. One half were transferred to Schenk &Hildebrandt+antibiotics and the other half directly toBoi2Y+antibiotics. For the first half, after 21 days on SH+antibioticsembryogenic calluses were transferred to Boi2Y+antibiotics where embryoswere allowed to develop and mature. For regeneration, torpedo-shapeembryos were removed from both set of calluses as soon as they appearedand placed on growth regulator-free MS without antibiotics for rooting.At the first appearance of root formation, plantlets were transferred toMS+IBA and then allowed to develop to mature plants. All the in vitromaterial was kept at 23° C./19° C. day-night T°, 16-h photoperiod, 16umol m⁻²s⁻¹ light irradiance.

Other protocols are also set out in Methods in Plant Molecular Biologyand Biotechnology pp. 67-76, Glick, Thompson (Eds) Library of Congress(1993). Further discussion of Agrobacterium transformation of alflafa isfound at An, G. Plant Physiol 81:86-91 (1986); Hoekema, A. et al Nature303:179-180 (1983); Chapter by Melton et al. in Alfalfa and AlfalfaImprovements Hanson, A. A., Barnes, D. K. and Hill, R. R., Jr. (Eds)pp.595-620 (1988); Madison, N. et al. Plant Cell Tiss. Org. Cult.42:255-260 (1995); and Shahin, E. A. et al. Crop Sci. 26:1235-1239(1986).

Regeneration

Culture Media

The following describes media used in regeneration of transformed andnon-transformed alfalfa. It is to be understood that those skilled inthe art could use media which varies considerably from these media andfall within the scope of the invention. The description is given by wayof example.

Gamborg's Based Medium

Gamborg's B-S medium is a widely used medium for culture of plantspecies. It is well known to those skilled in the art and is describedin detail at O. L. Gamborg, R. A. Miller, K. Ojima, Exp. Cell. Res.50:151-158 (1968). It forms a component of media listed below.

Modified B5 Medium

This medium is described at Atanassov, A. and Brown, D. C. W. Plant CellTissue and Organ Culture 3: 149-162 (1984). A typical mixture is thatformulated by GIBCO Laboratories and include: 1 mg/l 2,4-D, 0.2 mg/lkinetin, 30 g/l sucrose, 3000 mg/l KNO₃, 895 mg/l CaCl₂, 800 mg/l1-glutamine, 500 mg/l MgSO₄7H₂0, 100 mg/l serine, 10 mg/l L-glutathione,1 mg/l adenine, with the modification that was used instead of gelritereported in Atanassov, 9 g/l bacto agar. It forms a component of medialisted below.

MS Medium

This medium is well known to those skilled in the art and is describedin detail at T. Murashige and F. Skoog, Physiologia Plantarum 15:473-497(1962). A typical mixture is that formulated by Gibco Lab and includes:

Component mg/L NH₄NO³ 1650.0 KNO₃ 1900.0 CaCl₂ 2H₂O^(a) 440.0 MgSO₄7H₂O^(b) 370.0 KH₂ PO₄ 170.0 Na₂ EDTA 37.3 FeSO₄ 7H₂O 27.8 H₃ BO₃ 6.2MnSO H₂O 16.9 ZnSO₄ 7H₂O 8.6 K1 0.83 Na₂MoO₄..2H₂O 0.25 CuSO₄ 5H₂O 0.025CoCl₂ 6H₂O 0.025

Blaydes Medium and Modifications

This well known medium to those skilled in the art is described indetail at D. F. Blaydes, Physiol. Plant. 19:748-753 (1966).

BO (basal Blaydes medium) contains per liter: 300 mg KH₂P0₄, 100 mgKNO₃, 1 g NH₄NO₃, 347 mg Ca(N0₃)₂.4 H₂0, 35 mg MgSO₄.7 H₂0, 65 mg KCl,0.8 mg KI, 1.5 mg ZnSO₄.7 H₂ 0. 1.6 mg H₃B0₃, 4.4 mg MnSO₄H₂0, 2 mgglycine, 0.1 mg thiamine hydrochloride, 30 g sucrose, 10 g (5.57 gFeSO₄7H₂0 in 500 ml hot distilled water with 7.45 g Na2EDTA in 500 mlhot distilled water with pH to 5.9-6.0.

BII medium is the same as BO, but contains 2 mg/l each 15 NAA, Kinetin,and 2,4-D.

BOi2Y is the same as BO, but contains 100 mg/l inositiol and 2 g/l bactoyeast extract. After embryo induction, explants must be removed fromexposure to 2,4-D. 2,4-D appears to inhibit embryo development.

Schenk and Hildebrandt (SH) Medium

This medium is well known to those skilled in the art and is describedin detail at B. V. Schenk and A. C. Hildebrandt, Can. J. Bot. 50:199-204(1975). SHII contains 9.05 uM 2,4-dichlorophenoxy acetic acid (2,4-D)and 9.30 uM kinetin.

Modified SH Medium

This medium is well known to those skilled in the art and is describedin detail at D. H. Mitten, S. J. Sato, and T. A. Skokut, Crop Sci.24:943-945 (1984).

Modified SH medium contains: 25 uM α-naphthaleneacetic acid (NAA) and 10uM kinetin, callus was transferred to SE medium containing 50 uM 2,4-Dand 5 uM kinetin, transferred 3 days later to regeneration mediumcontaining BOi2Y.

The following is presented merely as examples and are not intended tolimit the scope of the invention. In each of the experiments set forthbelow, Regen-S, as described above, was employed. This variety is knownfor its high regeneration potential. Genes encoding the Alfalfa MosaicVirus coat protein (AMVcp), Phosphinotricin Acetyl Transferase (referredto here as BAR), Neomycin Phosphotransferase (NPTII) and β-glucuronidase(GUS), were transformed into this genotype using a DuPont PDS 1000A ^(˜)particle gun. The alfalfa mosaic virus coat protein may protect plantsfrom AMV pathogens, BAR inactivates the nonselective herbicidephosphinotricin, present in Basta medium and NPTII inactivateskanamycin. Plasmid pPHI251 encoding for NPTII, and AMVcp was used. A mapof this plasmid is shown in FIG. 1. Plasmid pPHI256 was separately usedas indicated below in coding for BAR, AMVcp, and GUS. A map of thisplasmid is found at FIG. 2.

EXPERIMENT 1

Alfalfa Mature Cotyledon Particle Gun Transformation on Basta® Selection

Explant: Mature Cotyledons of RegenS

Plasmid pPHI256 (GUS, AMVcp, BAR)

Bombardment: 8 cotyledons per plate (8 plates) bombarded twice with 1.8um tungsten particles

Culture: Seed germinated 2 days and embryonic axis removed fromcotyledon

Cotyledon plated to filters soaked with 0.25 M sorbitol and adaxialsurface bombarded twice

Cultured on modified B5 medium 2 days

3 day post-bombardment cotyledons cultured on a modified as mediumcontaining 2.5 mgAl Basta® for 9 wks

4 wks callusing/embryogenesis (B5 base, 1 mg/l 2,4-D and 0.2 mg/lkinetin)

2 wks embryogeny/embryo development (BS base, 0. 1 mg/l NAA)

3 wks embryo maturation (Boi2Y base, no hormones)

Rooted on 5 mg/l Basta®

Shoot tips cultures initiated

Results: 60 embryos recovered

11 browned and died during selection

10 abnormal sacrificed for GUS histochemical

staining (all negative)

31 abnormal recultured for callus (also GUS negative)

8 normal—5 survived higher selection

In this experiment, five plants were recovered from culture of bombardedmature cotyledons on modified B5 media containing 2.5 mg/l Basta®. Eachplant was identified to contain the AMVcp and BAR genes by the method ofpolymerase chain reaction amplification, as shown in Table 1.β-glucuronidase enzyme activity was also identified in the five plantsby a GUS assay described by Rao, G. and Flynn, P., BioTechniques, Vol.8, No. 1, pp. 38-40 (1990).

TABLE 1 Alfalfa Plants Recovered on Basta ® Selection GUS^(a) Shoot RootPCR Assay Assay Assay Assay Plant AMVcp^(b) BAR^(c) 1 2 1 2 E1 + + 3 — 22 E2 + + — — 2 1 E3 + + — 1 — N/A E4 + + 2 — — N/A E5 + + — — — —^(a)Fluorometric GUS assay expressed as pgn/ug total protein.^(b)Oligonucleotides target internal to AMVcp coding region.^(c)Oligonucleotides target CaMV promoter and 5′ region of BAR codingregion.

Below, PCR analysis of the parent and progeny is set 40 forth showing50% were positive for BAR. The first three plants are progeny followedby a maternal plant showing BAR expression, a paternal negative control,maternal plant positive for BAR and controls.

TABLE 2 PCR Analysis of Parent and Progeny Plants Sample Source BAR AMVBOO1E2 × YAE92 Progeny + − BOO1E2 × YA£9 Progeny − − BOO1E3 × YAE9Progeny − − Maternal BOO1E2 Maternal + − YAE92 Paternal Paternal − −Maternal BOO1E3 Maternal + − RA3 11-5 + control^(a) NPTII+ AMV+ − + RA3C308 − control − − ^(a)A description of this positive control is foundat Hill, et al., Bio/Technology, 9:373-377 (1991)

Southern analysis was performed on the parent plants which were found tobe clones and were positive by PCR for BAR and AMVcp genes. Thus, it canbe seen heritable transformation of plants was achieved.

In summary, it can be seen that transformation of mature cotyledons fromalfalfa can be accomplished through he use of microprojectilebombardment. However, as noted, regeneration is typically poor.Regeneration is dramatically improved by the use of immature cotyledonsin transformation and regeneration.

EXPERIMENT 2 Alfalfa Transformation using Agrobacterium

Agrobacterium was used to transform alfalfa, using mature and immaturecotyledons. Three different Agrobacterium tumefaciens strains were usedthat represented the three primary types of Agrobacterium strains:agropine, octopine, and nopaline. The Agrobacterium tumefaciens strainsused for transformation were A28 1 (An, G. supra), LBA4404 (Hoekema etal., Nature 303:179-180 (1983), and C58 (Zambryski et al., EMBO Journal2:2143-2150 (1983). The expression vectors were pGA643 (An, G. et al.Plant Mol. Biol. Manual A3:1-19 (1988) Kulwer Acad. Pub., Dordrecht) andpBibKan (Becker, Nucleic Acids Research 18:203 (1990)). AllAgrobacterium tumefaciens transformation strains were cultured,conjugated and maintained according to Desgagenes et al, Plant CellTiss. Org. Cult. 42:129-140 (1995).

Plants were established, crossed and tested from populations (genotypes,not preselected lines) of Regen S, Norseman, Cody, Iroquis, Ramsey, andMesa Sirsa. Mature and immature seed (10-15 days post pollination) wereharvested. Cotyledons were dissected as described herein. Eachstrain/vector combination was used for the transformation of 30 immaturecotyledons and 30 mature cotyledons. Preparation of Agrobacterium wasaccording to Chabaud et al., supra and Desgagnes, supra. Inoculum (OD600 nm between 0.18 and 0.20) were grown overnight in 10 ml liquid LB byshaking at 250 rpm at 28° C. The cultures were pelleted at 2300 ×g for15 mm., resuspended in the same volume of 0.85% saline, and diluted to aconcentration of 5×10⁷ bacteria/ml. Mature and immature cotyledons werewounded using a #11 scalpel blade. Cotyeldons were immersed for 10 mm inthe diluted bacteria, blotted between sterile filter papers, and placedon modified B5 medium for co-cultivation for 96 hours. Afterco-cultivation, explants were washed with sterile distilled water toremove excess bacteria, blotted and transferred to a selective modifiedB5 medium that also contained 50 μg/ml kanamycin and 300 μg/mlcarbenicillin for 28-40 days.

Calli transformation frequency was scored by the number ofkanamycin-resistant calli divided by the number of explants tested at 28days. Regeneration ability was scored by the appearance of somaticembryos from kanamycin resistant calli from 28-40 days postco-appearance of somatic embryos from kanamycin resistant calli from28-40 days post co-cultivation. Enzymatic analysis for neomycinphosphotransferase activity and amplification of the kanamycinresistance gene by the polymerase chain reaction was used to confirm thepresence and function of the transgene.

The table below sets forth transformation frequency of Kanamycinresistant calli (calli obtained/30 cotyledons plated)

TABLE 3 Transformation frequency of kanamycin resistant calli (calliobtained/30 cotyledons plated) Alfalfa genotype Strain/vector ImmatureCotyledons Mature Cotyledons Regen S C58/pGA643 0.47 0.33 C58/pBib-Kan0.63 0.50 A281/pGA643 0.33 0.40 A281/pBib-Kan 0.40 0.37 LBA 4404/pGA6430.27 0.30 LBA 4404/pBib-Kan 0.43 0.73 Norseman C58/pGA643 0.77 0.50C58/pBib-Kan 0.43 0.40 A281/pGA643 0.37 0.27 A281/pSib-Kan 0.23 0.43 LBA4404/pGA643 0.43 0.50 LBA 4404/pBib-Kan 0.30 0.17 Cody C58/pGA643 0.530.33 C58/pBib-Kan 0.27 0.40 A281/pGA643 0.20 0.13 A281/pBib-Kan 0.530.37 LBA 4404/pGA643 0.47 0.60 LBA 4404/pBib-Kan 0.20 0.33 IroquoisC58/pGA643 0.57 0.33 C58/pBib-Kan 0.30 0.47 A281/pGA643 0.10 0.23A281/pBib-Kan 0.47 0.40 LBA 4404/pGA643 0.57 0.23 LBA 4404/pSib-Kan 0.430.63 Ramsey C58/pGA643 0.13 0.30 C58/pBib-Kan 0.53 0.43 A281/pGA643 0.230.17 A281/pBib-Kan 0.27 0.40 LBA 4404/pGA643 0.47 0.23 LBA 4404/pBib-Kan0.30 0.63 Mesa Sirsa C58/pGA643 0.67 0.53 C58/pBib-Kan 0.33 0.20A281/pGA643 0.37 0.57 A281/pBip-Kan 0.10 0.33 LBA 4404/pGA643 0.53 0.50LBA 4404/pBib-Kan — 0.33

Production of transformed calli shows no statistically significantdifferences were found between use of immature or mature cotyledons forthe formation of kanamycin resistant calli.

Table 4 shows regeneration frequency of kanamycin resistant calli thatproduced somatic embryos.

TABLE 4 Regeneration frequency of kanamycin resistant calli thatproduced somatic embryos Alfalfa genotype Strain/vector ImmatureCotyledons Mature Cotyledons Regen S C58/pGA643 0.82 0.55 C58/pBib-Kan0.79 0.71 A281/pGA643 0.66 0.64 A281/pBib-Kan 0.64 0.60 LBA 4404/pGA6430.71 0.62 LBA 4404/pSib-Kan 0.83 0.73 Norseman C58/pGA643 0.35 0.36C58/pBib-Kan 0.50 0.27 A281/pGA643 0.30 0.00 A281/pBib-Kan 0.00 0.33 LBA4404/pGA643 0.42 0.21 LBA 4404/pBib-Kan 0.12 0.00 Cody C58/pGA643 0.370.00 C58/pBib-Kan 0.14 0.00 A281/pGA643 0.20 0.00 A281/pBib-Kan 0.190.00 LBA 4404/pGA643 0.31 0.00 LBA 4404/pBib-Kan 0.00 0.00 IroquisC58/pGA643 0.29 0.00 C58/pBib-Kan 0.37 0.00 A281/pGA643 0.00 0.00A281/pBib-Kan 0.31 0.00 LBA 4404/pGA643 0.23 0.00 LBA 4404/pBib-Kan 0.420.00 Ramsey C58/pGA643 0.00 0.00 C58/pBib-Kan 0.31 0.00 A281/pGA643 0.000.00 A281/pBib-Kan 0.14 0.00 LBA 4404/pGA643 0.23 0.00 LBA 4404/pBib-Kan0.13 0.00 Mesa Sirsa C58/pGA643 0.20 0.00 C58/pBib-Kan 0.33 0.00A281/pGA643 0.10 0.00 A281/pBib-Kan 0.00 0.00 LBA 4404/pGA643 0.27 0.00LBA 4404/pBib-Kan — 0.00

Regeneration of somatic embryos from kanamycin resistant calli asobserved in immature and mature explants in the two control populations(RegenS and Norseman); however, only immature explants gave rise tosomatic embryos from kanamycin resistant calli in elite alfalfavarieties.

Thus it is evident that the source of explant was key in obtainingregeneration of tissue transformed using the Agrobacterium method. It ispossible through the use of this invention to transform elite varietiesusing the Agrobacterium method which could not previously be transformedand regenerated.

Immature Cotyledons

Somatic embryogenesis can be direct, where embryos are formed directlyfrom the cells, or indirect where a callus is formed which goes throughde-differentiation. Where in the past research has centered on using aparticular germ plasma source, selecting for genotypes with improvedregeneration, recurrent selection to create varieties having improvedregeneration, or selection for genes in plant breeding techniques indeveloping improved regeneration lines, this invention uses an entirelydifferent approach. See, e g. Mitten, et al., Crop Science, 24:943(1984); Seitz, Kris & Bingham, In Vitro, 24:1047 (1988); Brown andAtanassov, Plant Cell Tissue Organ Culture, 4:111-122 (1985). Thus, theinvention relates to the use of immature cotyledons to improveregeneration, and thereby transformation of alfalfa.

The use of immature cotyledons has been found to be an important factorin regeneration. As a seed develops, from about 0-5 days pastpollination the seed embryo is globular in shape and generally withoutform, translucent in color. At about 5 days it demonstrates a heartshaped appearance. The embryo then undergoes rotation, and at about 10days has a visible cotyledon. The color is translucent to light green,and a scalpel placed behind the cotyledon can almost be visualized. Atabout 15 days the differentiation of the seed parts has become moredistinct, and by 20 days it has a dark green appearance. Beyond 25 days,the dark green color gives away to a yellowing. At 30 days it is creamywhite in color. It is at this point that the dormancy process isunderway.

It has been found by the applicant that immature cotyledons providingimproved regeneration include those which are formed up to 25 days pastpollination. At 5-7 days post-pollination the heart stage is apparent,however, as a practical matter it is difficult to excise the cotyledonportion at this stage and to differentiate it from the other parts ofthe embryo. The cotyledon can be harvested more easily beginning atabout 10 days when it has a translucent to very light green color. Thetime period between 10-IS days is preferred and provides forconsiderably improved regeneration results. The most preferable time toexcise the cotyledon is at about 10 days past pollination and/or thecotyledon has a translucent to light green color. The light green colorcan be compared to that found at Panton Color Chart No. PM5372.

As a result of using immature cotyledons as provided herein, it ispossible to regenerate varieties which have never been capable oftransformation and regeneration before.

Thus, while highly regenerable plants in the past have not alwayscarried the preferred phenotypes, now one may regenerate even elitelines of alfalfa. These elite lines typically have desirable productionqualities but notoriously poor regeneration.

As a further result, when immature cotyledons are used, one can obtaintransformation of such elite lines which could not be regeneratedpreviously after introduction of DNA. The mode of transformation is nolonger limiting and transformation may occur by bombardment or thepreviously known use of Agrobacteria, or other known methods, withregeneration now possible.

EXPERIMENT 3 Regeneration

The typical protocol for regeneration includes placing the immaturecotyledon explant on a modified B medium. After 21-28 days somaticembryos are transferred to MS medium and allowed to mature. Obviouslythere are a number of variations on this protocol known to those skilledin the art and this is given by way of example. The following showsimproved regeneration which correlates to explant age.

Plants from two varieties were divided into three groups. Six plantsfrom YAE92 were placed into a first group, five plants from YAE92 wereplaced into a second group, and five plants from YAM93 were placed intoa third group. Table 5 below shows the background of each variety. Eachgroup was crossed exclusively within itself from the resulting plants,each raceme is individually identified and its integrity maintained.Harvesting occurs at timed intervals from 0-30 days past pollination,with an early harvest from a numbered raceme and later harvest from thesame raceme. By maintaining the integrity of the group and harvestingfrom a numbered raceme over the time course of the experiment, it can bedemonstrated that variation of genotype even within a particular varietydoes not affect regeneration as long as regeneration is from immaturecotyledon. Each of the cotyledons excised at the time course harvest wasregenerated. A graph at FIG. 3 of the results plots the age of thecotyledon post-pollination on the x-axis and the regeneration responseon the y-axis. The results show that even from the same raceme there isincreasing regeneration beginning at just after pollination, up to about15 days past pollination, with declining regeneration up to maturity.

The scoring and evaluation of the time course harvest is shown in Table6 Thus, it is clear that age of the cotyledon excised is the criticalfactor effecting regeneration.

TABLE 5 Percent Contribution of Germplasm varia ladak turk falc chilperu indian african flemish unk. YAE 92 27 8 4 6 8 — — — 47 — YAM93 23 810 8 7 2 — — 42 —

TABLE 6 Regeneration Shown as Percent Response, From Immature Cotyledonsof Different Ages From Controlled Matings Within Three Groups of AlfalfaPlants AGE Number (Days Post- Cotyledons Percent Pollination) EvaluatedResponse 6 44 48 7 38 53 8 42 52 9 39 64 10 52 60 11 44 61 12 51 57 1353 62 14 38 55 15 42 43 16 38 34 17 42 27 18 49 22 19 59 17 20 56 14 2130 7 22 45 9 23 41 7 24 19 5 25 68 1 26 73 1 27 18 0 28 9 0 29 17 0 3017 0 31 15 0 32 11 0 33 15 0 34 9 0 35 10 0 36 17 0 37 18 0 38 14 0 3911 0 40 12 0

Thus, it can be seen that when immature cotyledons are used inregeneration of alfalfa, dramatically improved results occur.

EXPERIMENT 4

This experiment confirms that it is the immature cotyledon use whichprovides for the improved regeneration and may be applied to anygermplasm source. A number of varieties, including those that have pooror little regeneration were regenerated using immature cotyledons. Aminimum of twelve plants of each of the varieties listed in Table 5 wereplanted and pollinated, with the exception that 15 plants of Grimm (Pi452472), 30 plants of Mesa Sirsa and 1 plant of RA3 clone were plantedand pollinated. Each raceme identified was harvested at about 10-15 dayspast pollination and at maturity (about 30 days). Immature and maturecotyledons were regenerated as described in Experiment 23. The data inTable 7 below demonstrates that use of immature cotyledons substantiallyimproves regeneration even in those varieties which traditionally havepoor or no varieties that are extremely difficult to regenerate.Selected varieties and, in particular, those with the worstregeneration, are shown in terms of percent regeneration of maturecotyledons in the solid bar; and percent regeneration of immaturecotyledons, represented by the hashed bar. Use of immature cotyledonsresulted in improved regeneration in each instance, including thosevarieties with no regeneration using mature cotyledons.

TABLE 7 Comparison of Percent of Regeneration of 30 Days PastPollination Mature Cotyledons With Percent Regeneration of 10-15 DayPost-Pollination Immature Cotyledons # Mature % Mature # ImmatureImmature Alfalfa cotyledons cotyledons cotyledons cotyledons designationsampled regenerating sampled regenerating Grimm 206 0 223 15 (Pi 452472)Norseman 52 28 198 37 Lahontan 67 2 184 30 Turki stan 76 8 186 18 (Pi86696) Teton 145 3 175 16 Pi251689 140 0 129 21 Caliverde 65 138 0 16727 Buffalo 127 1 158 20 Cody 161 0 183 31 Hairy Peruvian 147 4 166 18Hairy Peruvian 150 0 173 22 (BIG-PLH) Mesa Sirsa 243 0 262 17 Sonora 11011 127 25 DuPuits 138 6 145 24 Iroquois 143 0 158 26 Vernal 152 22 16134 Culver 170 0 173 23 Agate 135 0 155 19 Ramsey 121 0 181 24 El Unico149 0 190 28 RegenS/RA 343 54 63 72 YAM93 164 0 196 34 YAE92 179 0 18727

EXPERIMENT 5

Three separate tests were conducted to determine if immature embryoscould be transformed.

In the first test, cotyledons were bombarded with pPHI413 (see FIG. 5),as above, and levels of GUS expression assayed. Forty-two samples werebombarded. Optimum expression occurred 48 to 72 hours post bombardmentwhere 26 of the 42 samples expressed GUS with a mean of 1.7 pg/pg totalprotein. Five days post bombardment 6 of 30 samples showed an average of2 pg/μg total protein, while at 17 days post bombardment 3 of 30 samplesshowed an average of 2 pg/μg total protein.

In the second, the effect of bombardment on alfalfa regeneration underselection was studied. Immature cotyledons of Regen S were harvested 11days post-pollination. Cotyledons were excised from the embryo,bombarded three times with the plasmid pPHI251 (FIG. 1), adsorbed totungsten particles, and cultured on modified 35 media containing 25 mg/lkanamycin sulfate. Somatic embryos were harvested approximately twomonths after treatment, allowed to desiccate on MS media for two months,and germinated on MS media containing 100 mg/l kanamycin sulfate. Leaftissue was harvested and assayed for neomycin phosphotransferase (NPTII)activity. The results are shown in Table 8.

TABLE 8 Plant NPTII activity pg/μg total protein (elisa) AMVcp CBX1063 + CBX107 2 − CBY107 5 − CBY108 4 − CBZ108 1 − CBX112 1 + CBY112 3 −CBZ112 1 − CBA112 1 − CBX115 2 − CBX116 2 + CBY116 3 − CBX117 3 − 1Regen S 3-11 13 − 2 Regen S 3-11 10 − 3 Regen S 3-11 9 − Regen SNegative Control^(a) 0 − Rambler Positive Control^(b) 4 + ^(a)Thenegative control was bombarded with TE buffer-treated tungsten particlesand regenerated on media not containing kanamycin. ^(b)Rambler PositiveControl was a previously identified transgenic alfalfa plant shown tocontain and express the neomycin phosphotransferase gene (Hill et al.,Bio/Technology, 9:373-377 (1991)).

In the third test, yet another embodiment of the invention isdemonstrated and the affect of bombardment on the regeneration oftransformed elite alfalfa varieties was examined. Immature cotyledonswere excised from 11 day post pollination embryos. Somatic embryos wereregenerated. Somatic embryos were bombarded five times with tungstenparticles adsorbed with the plasmid pPHI251 (FIG. 1) and cultured onmodified B5 media containing 25 mg/l kanamycin sulfate. Embryos weresubcultured at 20 days post-bombardment to fresh modified BS mediacontaining 25 mg/l kanamycin sulfate. Green somatic embryos wereharvested 50 days post bombardment and matured on MS medium containing100 mg/l kanamycin sulfate. Leaf samples were taken at 80 dayspost-bombardment and assayed for neomycin phosphotransferase activity.The results are shown in Table 9.

TABLE 9 Yam93 Regenerant (pg/μg Total Protein) NPTII Activity CB93.1 11CB93.2 13 CB93.3 3 CB93.4 8 CB93.5 4 C393.6 9 Yam93 negative control^(a)0 Ramble 10-1-1^(b) 2 ^(a)The negative control plant was regeneratedfrom bombarded immature cotyledons bombarded with TE-buffer treatedtungsten particles. ^(b)rambler 10-1-1 was a previously identifiedtransgenic plant shown to contain and express the neomycinphosphotransferase gene [Hill, et al., Bio/technology, 9:373-377(1991)].

The latter test demonstrates that when immature cotyledons are used toform somatic embryos, and then those embryos are bombarded, even moreplants are recovered. Furthermore, the resulting plant has been found toretain this ability to regenerate. Elite varieties can not only beregenerated, but also retain this property.

It can further be seen that bombardment of the immature embryos orsomatic embryos does not adversely affect regeneration and that DNA isexpressed in these now regenerable cells and plants.

The foregoing demonstrates transformation of Medicago sativa,transformation with particle acceleration, and that substantiallyimproved regeneration of Medicago sativa is possible by the use ofimmature cotyledons. Regeneration of varieties not previouslyregenerated or with very poor regeneration is achieved. Thus,transformation of these same varieties is now possible.

Thus, it can be seen the invention accomplishes its objectives.

What is claimed is:
 1. A process of producing a genetically transformedalfalfa plant comprising introducing heterologous nucleotide sequencesinto cells of immature cotyledons of alfalfa and cultivating the cellswith the heterologous nucleotide sequences into alfalfa plants.
 2. Theprocess of claim 1 wherein the immature cotyledons are six to 25 dayspast pollination.
 3. The process of claim 1 wherein the immaturecotyledons are 10 to 15 days past pollination.
 4. The process of claim 1wherein the immature cotyledons are translucent to light green in color.5. The process of claim 1 wherein the nucleotide sequence is introducedinto elite varieties of alfalfa.
 6. The process of claim 1 wherein theheterologous nucleotide sequence is prepared as a plasmid, hosted inAgrobacterium tumefaciens and combined with the immature cotyledonalfalfa cells such that the heterologous nucleotide sequence isintroduced into the alfalfa cells.